In vitro cleavage of avian retrovirus gag proteins by viral protease p15.
In vitro cleavage of avian retrovirus gag proteins by viral protease p15.
复制标题
病毒蛋白酶 p15 对禽逆转录病毒 gag 蛋白的体外裂解。
DOI：
10.1016/0042-6822(79)90534-8
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发表时间：
1979
期刊：
影响因子：
3.7
通讯作者：
R. Eisenman
中科院分区：
文献类型：
--
作者：
V. Vogt;A. Wight;R. Eisenman
禽成髓细胞瘤病毒含有蛋白水解活性，可以在体外裂解病毒前体多肽Pr76gag。该底物通过体内放射性标记制备，随后进行免疫沉淀、十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳以及从凝胶中洗脱。该反应的主要产物包括成熟的病毒体蛋白 p27 和 p15，以及包含这两种蛋白的不稳定片段。还形成了其他几个片段，但成熟的 p12 和主要的 p19 物种没有形成。与抗体和福尔马林固定的葡萄球菌结合的未变性 Pr76 的裂解产生类似的片段。病毒蛋白水解酶与结构蛋白 p15 没有区别。 p15 对 Pr76 的裂解在 pH 范围 4-7 范围内是最佳的，并且受到盐的刺激。该酶的活性不会被活性位点具有丝氨酸的蛋白酶特异性试剂抑制，但会被硫醇特异性试剂部分抑制。蛋白水解具有高度特异性。在用于 Pr76 裂解的条件下，p15 不会在与 Pr76 平行从含有 SDS 的凝胶中洗脱的细胞蛋白混合物中引入断裂。然而，它会片段化含有全部或部分 Pr76 氨基酸序列的蛋白质。这些蛋白质包括病毒逆转录酶的前体多肽 (Pr180gag-pol)、未感染的 gs+chick 细胞 (P120) 中发现的病毒相关蛋白质、来自感染禽类成红细胞增多症病毒 (P75) 或禽类骨髓细胞瘤病毒 MC29 (P110) 的细胞的病毒蛋白。
Avian myeloblastosis virus contains a proteolytic activity that can cleavein vitrothe viral precursor polypeptide Pr76gag. This substrate was prepared by radioactive labelingin vivofollowed by immune precipitation, polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate, and elution from the gel. The major products of this reaction include the mature virion proteins p27 and p15, as well as an unstable fragment containing both of these proteins. Several other fragments are also formed, but mature p12 and the major p19 species are not. The cleavage of undenatured Pr76 bound to antibodies and formallin-fixedStaphylococcusyields similar fragments. The viral proteolytic enzyme is indistinguishable from the structural protein p15. Cleavage of Pr76 by p15 is optimal in the pH range 4–7 and is stimulated by salt. The activity of the enzyme is not inhibited by reagents specific for proteases with serine at their active sites, but is partially inhibited by reagents specific for thiols. Proteolysis is highly specific. Under the conditions used for Pr76 cleavage, p15 does not introduce breaks into mixtures of cellular proteins eluted in parallel to Pr76 from SDS-containing gels. However, it does fragment proteins that contain all or parts of the amino acid sequence of Pr76. These proteins include the precursor polypeptide for viral reverse transcriptase (Pr180gag-pol), a virus-related protein found in uninfected gs+chick cells (P120), viral proteins from cells infected with avian erythroblastosis virus (P75) or with avian myelocytomatosis virus MC29 (P110).